The biochemical pathway by which the DNA of bacteriophage lambda integrates into its E. coli host has been investigated. We analyzed features of attP, the DNA segment carried by the virus that is essential for integrative recombination. First, we determined the role within attP of three binding sites for an E. coli protein, IHF, by making oligonucleotide-directed mutations that inactivate each site. All three sites proved to be essential for efficient integration but subtle differences between the behavior of the mutants in vivo suggest different roles for the different sites. Second, we developed a method for studying the interaction of specific binding proteins with supertwisted DNA. We used this new DNA footprinting method to show that Int, a viral recombinase, and IHF cooperate to form a complex nucleoprotein structure at attP only when it is supercoiled. The degree and sign of supercoiling needed to generate the structure correlate well with those needed to promote integration. Finally, we used chemical and enzymatic DNA synthesis to construct analogs of the bacterial recombination site, attB, the contain phosphorothioates in place of critical phosphate residues in DNA. These analogs recombine poorly and accumulate Holliday structures, presumptive intermediates in recombination. We found that the formation of Holliday structures was independent of DNA homology between attB and attP but homology was essential to complete the recombination. This eliminates models that invoke homology for the synapsis step of recombination.